Method for AFM investigation of lateral forces required for the surface detachment of neurites

Resprouting of neurites to establish direct connections between stimulation electrodes and spiral ganglion neurons is of considerable interest in current cochlear implant research. The adhesion strength of neurites to implant surfaces is a critical factor determining whether such connections develop, remain stable or are disrupted shortly after formation. In this study, an in vitro protocol was established to quantify the lateral forces required to detach neurites from the surface of precoated substrates using atomic force microscopy. Spiral ganglion neurons were isolated from mixed primary cell cultures using cytosine β-D-arabinofuranoside hydrochloride as a mitotic inhibitor. After several days of cultivation, the neurites were mechanically hooked with the cantilever tip and laterally dragged across the substrate surface to measure the forces required for detachment. Four different parameters and their influence on the detachment process were evaluated: exposure time to measurement conditions, the distance between growth cone and cantilever, the distance between cantilever and soma, and the total length of the neurite. The mean lateral detachment force was 2.47 ± 0.19 nN, and no significant correlation was observed between the lateral forces and any of the four investigated parameters. Overall, this study provides quantitative information on neurite-surface adhesion behaviour and strength in vitro.